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PubMed1995

Persistence of protamine precursors in mature sperm nuclei of the mouse.

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a protamine of 106 residues (pmP2) with an amino-terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin. This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis.

Amino Acid Sequence↗
PubMed1995

Kit ligand mediates survival of type A spermatogonia and dividing spermatocytes in postnatal mouse testes.

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c-kit is expressed in type A, intermediate, and B spermatogonia, and kit-ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c-kit function. The present work was undertaken to determine whether blocking c-kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8-day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c-kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SId/+ males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15-fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output.

Animals↗
PubMed1989

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: evidence for translation during spermiogenesis.

An antiserum prepared against the purified protein carboxyl methyltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosis/atrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4-5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.

Aging↗
PubMed2005

DkkL1 (Soggy), a Dickkopf family member, localizes to the acrosome during mammalian spermatogenesis.

Dickkopf-like 1 (DkkL1) is related to the Dickkopf gene family, a group of proteins that are characterized as secreted antagonists of Wingless (Wnt) signal transduction proteins. DkkL1 mRNA is found in preimplantation mouse embryos and in developing neural tissue, but in adults it is found primarily in the testes. In an effort to elucidate its function, the distribution of DkkL1 protein in mouse testis and mature sperm was analyzed by immuno-histochemistry and immuno-blotting techniques. DkkL1 first appeared in the developing spermatocytes in seminiferous tubules as early as Stage XII, coincident with the appearance of DkkL1 mRNA. Surprisingly, however, DkkL1 localized to the developing acrosome in spermatocytes and spermatids and to the acrosome in mature sperm. Furthermore, DkkL1 was N-glycosylated in the testis, but it did not appear to be excreted, and the DkkL1 in mature sperm was no longer N-glycosylated, suggesting that additional post-translational modifications occurred during the final stages of spermatogenesis. These results identify a member of the Dickkopf family as a novel acrosomal protein that may be involved in acrosome assembly or function, a unique role for a secreted signaling molecule.

Acrosome↗
PubMed2006

Calpain 11 is unique to mouse spermatogenic cells.

The calpains are a family of calcium-dependent thiol proteases involved in intracellular processing of proteins. They occur as heterodimers containing one of various large subunits and a common small subunit. Some of the large subunits are expressed ubiquitously and others are expressed in a restricted set of tissues. We have cloned the cDNA for mouse calpain 11 and demonstrated that it is expressed specifically in the mouse testis. The mRNA begins to accumulate in the testis between days 14 and 16 after birth, corresponding to the period of pachytene spermatocyte development. The protein is detected by day 18 after birth, during mid to late pachytene spermatocyte development, and is present in the acrosomal region of spermatozoa from the cauda epididymis. The expression of calpain 11 during spermatogenesis and its localization in spermatozoa suggest that it is involved in regulating calcium-dependent signal transduction events during meiosis and sperm functional processes.

Amino Acid Sequence↗
PubMed2006

Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells.

Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.

Amino Acid Sequence↗
PubMed1986

Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol.

Pregnant female mice were given ethinyl estradiol on days 11 through 17 of gestation. On day 18 the dams were killed and the male fetuses were examined for testicular differentiation. Three of 12 males from dams treated with the highest dose of ethinyl estradiol showed cryptorchid testes with uterine tubes. Light and electron microscopic evaluation of the testes, both cryptorchid and normal, demonstrated foci of hyperplasia of Leydig cells showing cytoplasmic and nuclear pleomorphism, increase in lipid droplets, and decrease in smooth endoplasmic reticulum and ribosomes when compared to testes from control fetal mice. Morphometric determinations of the testes indicated that the number of Leydig cells in a unit area (mm2) in the interstitial tissue showed a dose-response relationship to ethinyl estradiol in the normal testes. The number of Leydig cells in the testes exposed to the highest dose of estrogen showed a significant difference between cryptorchid and normal testes: the former had fewer Leydig cells than the latter. These morphological observations indicate that hyperplasia of Leydig cells of fetal mouse testis at term can be induced by transplacental treatment with ethinyl estradiol and suggest that a malignant transformation into a Leydig cell tumor is possible.

Animals↗
PubMed1996

Cloning and expression of mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T3.

A novel isoform of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated ppGaNTase-T3, has been cloned from a mouse testis cDNA library and expressed in COS7 cells. ppGaNTase-T3 displayed 64 and 59% amino acid identity with ppGaNTase-T1 and ppGaNTase-T2, respectively, and 96% amino acid identity with the recently reported human form of ppGaNTase-T3. The ppGaNTase-T3 transcript is abundant in the major salivary glands, gastrointestinal tract and both the male and female reproductive systems. ppGaNTase-T3 and ppGaNTase-T1 display overlapping substrate preferences in vitro, although mapping studies of O-glycosylated peptides suggests that certain hydroxyamino acids are preferentially glycosylated by each isoform. This suggests that more than one isoform of ppGaNTase may be required to complete the O-glycosylation of endogenous substrates.

Amino Acid Sequence↗
PubMed1993

Binding of germ cells to mutant Sld Sertoli cells is defective and is rescued by expression of the transmembrane form of the c-kit ligand.

Mutations in the steel (Sl) locus, encoding the c-kit ligand (KL), are associated with impaired germ cell development in mice. Two forms of KL exist: one more steadily associated with the plasma membrane and one more easily released as a soluble protein. We report here that the expression of the two mRNAs coding for the two different form of KL is developmentally regulated in mouse testis. At birth the two mRNAs are expressed at an equal ratio. Starting after 6 days of life, and in parallel to initiation of germ cell differentiation, the mRNA encoding the membrane-associated form of KL becomes more abundant. Germ cells, and especially spermatogonia, express c-kit; thus membrane-bound KL could mediate adhesion between Sertoli cells and germ cells. We find, in fact, that Sertoli cells from Sl/Sld mutant mice, which do not express the mRNA for the membrane-associated form of KL, are unable to bind germ cells. Introduction of a plasmid expressing the transmembrane form, but not the soluble form, of KL restores the ability of Sertoli cells from Sl/Sld mutant mice to bind germ cells. These data suggest that preferential expression of the membrane-anchored form of KL in Sertoli cells may have a role in mediating adhesion of c-kit-expressing germ cells to Sertoli cells.

Aging↗
PubMed1998

Molecular cloning and characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein).

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

Amino Acid Sequence↗
PubMed1996

Localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study.

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42 degrees C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

Animals↗
PubMed1994

Chromosomal assignment of three novel mouse genes expressed in testicular cells.

The chromosomal positions of three genes that are selectively expressed in mouse testis cells have been identified. These genes include (i) TAZ83, which codes for an early- to mid-pachytene germ cell stage-expressed, cysteine-rich transmembrane protein (cyritestin) with homologies to various snake toxins and guinea pig sperm-egg fusion proteins; (ii) TNZ1, which is expressed in neonatal Leydig cells; and (iii) TAZ4, a testis-specific gene isolated by immunoscreening with antiserum raised against Sertoli cell membranes. Our experimental data, derived from chromosomal in situ hybridizations and RFLP studies of genetic backcrosses, indicate that (i) the TAZ83 (cyritestin) gene maps to chromosome 8, band A2, near the Plat locus; (ii) TNZ1 is located in the proximal region of chromosome 11; and (iii) TAZ4 is located at band D in the distal portion of chromosome 11, near the Hlr1 locus, with a related sequence, TAZ4-rs1, in the proximal part of chromosome 1.

Animals↗
PubMed1999

Characterization of the mouse Xpf DNA repair gene and differential expression during spermatogenesis.

The human XPF protein, an endonuclease subunit essential for DNA excision repair, may also function in homologous recombination. To investigate a possible link between mammalian XPF and recombination that occurs during meiosis, we isolated, characterized, and determined an expression profile for the mouse Xpf gene. The predicted mouse XPF protein, encoded by a 3.4-kb cDNA, contains 917 amino acids and is 86% identical to human XPF. Appreciable similarity also exists between mouse XPF and homologous proteins in budding yeast (Rad1), fission yeast (Rad16), and fruit fly (Mei-9), all of which have dual functions in excision repair and recombination. Sequence analysis of the 38.3-kb Xpf gene, localized to a region in proximal mouse chromosome 16, revealed greater than 72% identity to human XPF in 16 regions. Of these conserved elements, 11 were exons and 5 were noncoding sequence within introns. Xpf transcript and protein levels were specifically elevated in adult mouse testis. Moreover, increased levels of Xpf and Ercc1 mRNAs correlated with meiotic and early postmeiotic spermatogenic cells. These results support a distinct role for the XPF/ERCC1 junction-specific endonuclease during meiosis, most likely in the resolution of heteroduplex intermediates that arise during recombination.

Amino Acid Sequence↗
PubMed1999

USP25, a novel gene encoding a deubiquitinating enzyme, is located in the gene-poor region 21q11.2.

We have identified a new gene, USP25, spanning over 150 kb at 21q11. 2, one of the lowest gene-density regions of the human genome. USP25 is made up of 25 exons and encodes a 1087-aa protein. Database comparisons reveal high homology with members of the ubiquitin protease family (UBP). Basal expression was observed in all human tissues tested, and two main transcripts were identified. The homologous murine gene has also been characterized. In situ hybridization in mouse embryonic brains showed a clear correlation of expression with proliferative neuroepithelial cells and postmitotic neurons. Moreover, high expression was observed in adult mouse testis. UBPs belong to a complex family of deubiquitinating enzymes that specifically cleave ubiquitin conjugates on a great variety of substrates. These enzymes have an essential role in protein degradation via the 26S proteasome and thus regulate many cellular pathways. An increase in USP25 gene dosage in Down syndrome patients could seriously disturb the balance between ubiquitinated and deubiquitinated substrates.

Amino Acid Sequence↗
PubMed1996

Structural comparison of tektins and evidence for their determination of complex spacings in flagellar microtubules.

Recent structural studies indicate that a tektin heteropolymer forms a unique protofilament of flagellar microtubules. We report here the sequence of tektin C (approximately 47 kDa), predicted from its cDNA (GenBank U38523), compared to tektins A (approximately 53 kDa) and B (approximately 51 kDa) from sea urchin (Strongylocentrotus purpuratus) sperm flagellar microtubules, and compared to partial sequences reported from mouse and human. We are now able to make several observations concerning the tektin family: (1) their common structural features, (2) a comparison of their structure to intermediate filament proteins, and (3) their possible organization in the tektin filament polymer. The predicted amino acid sequence identities/similarities are: for tektins A and C, 42/54%, for tektins A and B, 34/51%; for tektins B and C, 29/42%; for tektin C and a partial cDNA clone from mouse testis, 55/65%; and for tektin B and a partial cDNA clone from the human brain, 45/47%. The three tektins (and the human clone) possess the exact sequence repeat RPNVELCRD. The structural pattern of all three tektin polypeptides is similar to intermediate filament proteins. Tektins are predicted to form extended rods composed of two alpha-helical segments (approximately 180 residues long) capable of forming coiled coils, which are interrupted by short non-helical linkers. The two segments are homologous in sequence and secondary structure, indicating a gene duplication event prior to the divergence of the three tektins. Along each tektin rod cysteine residues occur with a periodicity of approximately 8 nm, coincident with the axial repeat of tubulin dimers in microtubules. From EM data and calculations of secondary structure, the segment length of tektin AB heterodimers is likely to be 16 nm. Both segments of tektin C may be 24 nm long, but one may be 16 nm. On the basis of the available evidence, we propose that coassembly of tektin AB heterodimers with tektin C dimers produces filaments with overall repeats of 8, 16, 24, 32, 40, 48 and 96 nm, generating the basis for the complex spatial arrangements of axonemal components.

Amino Acid Sequence↗
PubMed1996

A general bacterial expression system for functional analysis of cDNA-encoded proteins.

A general system for functional analysis of cDNA-encoded proteins is described. The basic concept involves the expression in Escherichia coli of selected portions of cDNAs in an approach toward the understanding of the function of the corresponding proteins. A selected cDNA is expressed as part of a fusion protein used for immunization to elicit antibodies, and a corresponding fusion protein, having the cDNA-encoded portion in common, for purification of target protein-specific antibodies. This antiserum could be used for functional analysis of the cDNA-encoded protein, e.g., by immunohistology. Two general expression vector systems for E. coli have been constructed, both (i) designed with multiple cloning sites in three different reading frames, (ii) having their protein production controlled by the tightly regulated T7 promoter, and (iii) enabling affinity purification of the expressed target proteins by fusions to IgG-binding domains derived from staphylococcal protein A or a serum albumin-binding protein derived from streptococcal protein G, respectively. This novel system has been evaluated by expressing five cDNAs, isolated from prepubertal mouse testis by a differential cDNA library screening strategy. All five clones could be expressed intracellularly in E. coli as fusion proteins with high production levels, ranging from 4 to 500 mg/liter, and affinity purification yielded essentially full-length products. Characterization of affinity-purified antibodies revealed that there exists no cross-reactivity between the two fusion systems and that such antibodies indeed could be used for immunohistology. The implications for the described system for large-scale functional analysis of cDNA libraries are discussed.

Animals↗
PubMed1999

Involvement of germ cell apoptosis in the induction of testicular toxicity following hydroxyurea treatment.

The present study investigated the occurrence of apoptotic cell death in the mouse testis at various intervals following the administration of hydroxyurea (HU). The presence of apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and by DNA fragmentation assay using ligation-mediated polymerase chain reaction. Both the incidence of apoptotic cells and the level of DNA fragmentation in the testis increased depending on the HU dose, and they were most apparent at the highest dose (400 mg/kg). The incidence of apoptotic cells in the HU-treated group increased continuously and peaked at 12 h, but then decreased gradually, reaching control levels by 48 h. After HU treatment, TUNEL-positive apoptotic cells increased in the seminiferous epithelium of the tubules, and affected cells were found synchronously in the tubules of animals treated with HU. Spermatogonia and spermatocytes were found to be affected selectively. TUNEL-positive cells were found to be stage-specific and were primarily in stage IV-VI tubules. It has been shown that in vivo HU exposure induced testicular germ cell apoptosis dose dependently in a time- and stage-specific manner, and damaged cells appeared to be eliminated by phagocytosis by neighboring cells. Apoptosis of damaged testicular germ cells is apparently a common response to various testicular toxicants therefore protecting the next generations of germ cells from the damaged cell population.

Animals↗
PubMed1986

Isolation of transcribed DNA sequences from chromosome 21 using mouse fetal cDNA.

A technique for isolating DNA sequences that are likely to be transcriptionally active in both humans and mice has been developed. This method is based on the screening of a human genomic DNA library with single stranded cDNA prepared from late gestation mouse fetal RNA. Using a human chromosome 21 DNA library, we have isolated and characterized two clones which are entirely composed of single copy human DNA sequences and are 2.7 and 6.7 kilobases in length. The 2.7 kilobase clone is homologous to a transcript found predominantly in nonpolyadenylated human fibroblast RNA. It also shows homology to mouse genomic DNA and recognises several polyadenylated RNAs in mouse testis. This cloned sequence has been mapped by in situ hybridization to the distal third of chromosome 21, the region involved in the causation of Down syndrome.

Animals↗